Para-hydroxystyrene (pHS) is an aromatic compound that has potential utility in a wide variety of industrial applications. For example, pHS and its acetylated derivative, para-acetoxystyrene (pAS) have application as monomers for the production of resins, elastomers, adhesives, coatings, automotive finishes, inks and electronic materials, and additives in elastomer and resin formulations.
A number of methods for the chemical synthesis of pHS are known. For example, pHS may be produced from ethyl benzene in a five-step process (U.S. Pat. No. 4,503,271) or from para-hydroxyacetophenol in a two step process (U.S. Pat. No. 5,523,378). Although it is possible to generate pHS by these methods, they typically require strongly acidic or basic reaction conditions, high reaction temperature, and generate large amounts of unwanted byproducts. In addition, chemical methods require expensive starting materials, which raise the cost of producing pHS. Despite the wide variety of uses for pHS, an inexpensive source of the material has not been developed.
A biological process for the production of pHS from a simple carbon source such as glucose is described by Ben-Bassat et al. (U.S. Patent Application Publication No. 2004/0018600). In that disclosure, a recombinant host cell expressing at least one gene encoding a polypeptide having para-hydroxycinnamic acid decarboxylase (PDC) activity in combination with either at least one gene encoding a polypeptide having tyrosine ammonia lyase (TAL) activity or at least one gene encoding a polypeptide having phenylalanine ammonia lyase (PAL) activity is used to produce pHS. A PAL activity converts phenylalanine to para-hydroxycinnamic acid (PHCA) in the presence of a P-450/P-450 reductase [cinnamate-4-hydroxylase (C4H) and P-450 reductase] system. An enzyme having a high TAL activity converts tyrosine directly to pHCA without any intermediate steps. Then, para-hydroxycinnamic acid decarboxylase (PDC) converts the pHCA to pHS. However, a problem encountered with the biological production of pHS is end-product inhibition, which limits product yield. Specifically, the rate of production of the product by the microorganism decreases as the concentration of the product increases. Additionally, the PDC enzyme and the microorganism are inactivated by the product when a certain critical concentration is reached in the fermentation medium.
One approach to mitigate end-product inhibition by pHS is to use two-phase extractive fermentation, in which the pHS produced by a recombinant production host is extracted into an immiscible organic phase during the fermentation so that it never reaches an inhibitory or critical concentration, as described by Ben Bassat et al. in co pending U.S. Patent Application No. 60/462,827. The methods described in that disclosure resulted in improved yields for pHS. However, still higher yields are required for commercial applications.
Tetsuji et al. (JP 11187870) describe a method for producing pHS, having a deuterium atom at the vinyl position, from pHCA using PDC isolated from Klebsiella oxytoca. The decarboxylase reaction is carried out in an aqueous buffer containing deuterated water. Ago et al. in U.S. Pat. No. 5,955,137 describe a method for producing 4-vinylguaiacol (4-hydroxy-3-methoxystyrene), a derivative of pHS, from ferulic acid (4-hydroxy-3-methoxycinnamic acid) in aqueous buffer using an enzyme source having ferulic acid decarboxylase activity. The product yields in both these methods are limited by product inhibition of the enzyme. Additionally, the recovery of the product is complicated because the product must be isolated from the substrate, biocatalyst, and buffer salts.
A biocatalytic method for producing pHS from PHCA in a biphasic reaction medium would decouple the production of PHCA and pHS, thereby enabling the optimization of both processes independently. The use of a biphasic reaction medium, consisting of an aqueous phase and a water-immiscible organic phase, in biocatalytic reactions can provide both kinetic and thermodynamic advantages (Bruce et al., Biotechnol. Prog. 7:116-124 (1991)). With the proper choice of organic solvent, the product is continuously removed from the aqueous phase, thereby reducing product inhibition, resulting in higher product yields. Moreover, product recovery is greatly simplified because the product can be readily isolated from the organic phase. Lee et al. (Enzyme Microb. Technol. 23:261-266 (1998)) describe the production of 4-vinylguaiacol (4-hydroxy-3-methoxystyrene), a derivative of pHS, via the decarboxylation of ferulic acid by resting cells of Bacillus pumilus having ferulic acid decarboxylase activity using a two-phase, biocatalytic process. Several solvents were evaluated, including chloroform, methylene chloride, ethylacetate, ethyl ether, petroleum ether, cyclohexane, and C5-C8 alkanes. Hexane was selected as the preferred solvent. However, the production of pHS from pHCA using an enzyme source having PDC activity in a biphasic reaction medium is not described in that disclosure.
Therefore, the need exists for a method for producing pHS in high yield. The need also exists for a biocatalytic method for producing pHS in which the activity of the biocatalyst is preserved. It is also desirable to be able to reuse the biocatalyst over multiple reaction cycles for the method to be commercially viable.
Applicants have solved the stated problem by discovering a method for producing pHS in high yield using biocatalytic conversion of pHCA to pHS using an enzyme source having para-hydroxycinnamic acid decarboxylase activity in a biphasic reaction medium.